A. Viral Hemorrhagic Fever
Viral hemorrhagic fevers (VHFs) are a group of febrile illnesses caused by RNA viruses from several viral families. These highly infectious viruses lead to a potentially lethal disease syndrome characterized by fever, malaise, vomiting, mucosal and gastrointestinal (GI) bleeding, edema and hypotension. The four viral families known to cause VHF disease in humans include Arenaviridae, Bunyaviridae, Filoviridae and Flaviviridae.
In acute VHF, patients are extremely viremic, and mRNA evidence of multiple events cytokine activation exists. In vitro studies reveal these cytokines lead to shock and increased vascular permeability, the basic pathophysiologic processes most often seen with VHF. Multi-system organ failure affecting the hematopoietic, neurologic and pulmonary systems often accompanies the vascular involvement. Another prominent pathologic feature is pronounced macrophage involvement. Inadequate or delayed immune response to these novel viral antigens may lead to rapid development of overwhelming viremia. Extensive infection and necrosis of affected organs also are described. Hemorrhagic complications are multifactorial and are related to hepatic damage, consumptive coagulopathy and primary marrow injury to megakaryocytes. Aerosol transmission of some VHF viruses is reported among nonhuman primates and likely is a mode of transmission in patients with severe infection. Specific symptoms of VHF and modes of transmission vary depending on the particular viral pathogen.
B. Filoviruses
Filoviruses are enveloped viruses with a genome consisting of one linear single-stranded RNA segment of negative polarity. The viral genome encodes 7 proteins. Nucleoprotein (NP), virion protein 35 kDa (VP35) and virion protein 30 kDa (VP30) are associated with the viral ribonucleoprotein complex. VP35 is known to be required for virus replication and is thought to function as a polymerase cofactor. The viral RNA-dependent RNA polymerase is termed L (for large protein). The matrix protein (VP40) is the major protein of the viral capsid. The remaining proteins include virion glycoprotein (GP) and membrane-associated protein (VP24), which is thought to form ion channels. The Ebola viruses have one additional protein, small secreted glycoprotein (SGP).
Members of the filovirus genus include Zaire Ebola virus, Sudan Ebola virus, Reston Ebola virus, Cote d'Ivoire Ebola virus and Marburg virus. Ebola and Marburg viruses can cause severe hemorrhagic fever and have a high mortality rate. Ebola virus (Zaire and Sudan species) was first described in 1976 after outbreaks of a febrile, rapidly fatal hemorrhagic illness were reported along the Ebola River in Zaire (now the Democratic Republic of the Congo) and Sudan. Sporadic outbreaks have continued since that time, usually in isolated areas of central Africa. In 1995, eighteen years after the first outbreak was reported, Zaire Ebola reemerged in Kikwit, Zaire with 317 confirmed cases and an 81% mortality rate. The natural host for Ebola viruses is still unknown. Marburg virus, named after the German town where it was first reported in 1967, is primarily found in equatorial Africa. The host range of Marburg virus includes non-human and human primates. Marburg made its first appearance in Zimbabwe in 1975 and was later identified in other African countries, including Kenya (1980 & 1987) and Democratic Republic of the Congo (1999). Marburg hemorrhagic fever is characterized by fever, abdominal pain, hemorrhage, shock and a mortality rate of 25% or greater (“The Springer Index of Viruses,” pgs. 296-303, Tidona and Darai eds., 2001, Springer, N.Y.).
C. Flaviviruses
Flaviviridae is a family of viruses that includes the genera flavivirus, hepacivirus and pestivirus. Viruses in the genus flavivirus are known to cause VHFs. Flaviviruses are enveloped viruses with a genome consisting of one linear single-stranded RNA segment of positive polarity. The RNA genome has a single open reading frame and is translated as a polyprotein. The polyprotein is co- and post-transcriptionally cleaved by cell signal peptidase and the viral protease to generate individual viral proteins. Viral structural proteins include capsid (C), precursor to M (prM), minor envelope (M) and major envelope (E). Flavivirus non-structural proteins include NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5. NS1, NS2A, NS3 and NS4A are found in the viral replicase complex. In addition, NS3 is known to function as the viral protease, helicase and NTPase. NS2B is a co-factor for the protease function of NS3. NS5 is the viral RNA-dependent RNA polymerase and also has methyltransferase activity.
Members of the flavivirus genus include yellow fever virus, Apoi virus, Aroa virus, Bagaza virus, Banzi virus, Bouboui virus, Bukalasa bat virus, Cacipacore virus, Carey Island virus, Cowbone Ridge virus, Dakar bat virus, dengue virus, Edge Hill virus, Entebbe bat virus, Gadgets Gully virus, Ilheus virus, Israel turkey meningoencephalomyelitis virus, Japanese encephalitis virus, Jugra virus, Jutiapa virus, Kadam virus, Kedougou virus, Kokobera virus, Koutango virus, Kyasanur Forest disease virus, Langat virus, Louping ill virus, Meaban virus, Modoc virus, Montana myotis leukoencephalitis virus, Murray Valley encephalitis virus, Ntaya virus, Omsk hemorrhagic fever virus, Phnom Phenh bat virus, Powassan virus, Rio Bravo virus, Royal Farm virus, Saboya virus, Sal Vieja virus, San Perlita virus, Saumarez Reef virus, Sepik virus, St. Louis encephalitis virus, Tembusu virus, tick-borne encephalitis virus, Tyuleniy virus, Uganda S virus, Usutu virus, Wesselsbron virus, West Nile virus, Yaounde virus, Yokose virus, Zika virus, cell fusing agent virus and Tamana bat virus.
A number of flaviviruses cause human disease, particularly hemorrhagic fevers and encephalitis. Each species of flavivirus has a unique geographic distribution; however, taken together, flaviviruses, and flavivirus-induced disease, can be found world-wide. One of the more commonly known diseases is dengue fever, or dengue hemorrhagic fever/shock, which was first described as a virus-induced illness in 1960. Dengue fever occurs in tropical and temperate climates-and is spread by Aedes mosquitoes. The mortality rate is 1-10% and symptoms include febrile headache, joint pain, rash, capillary leakage, hemorrhage and shock. Another common flavivirus-induced disease is yellow fever. Yellow fever is found in tropical Africa and America and is transmitted by mosquitoes. The mortality rate is approximately 30% and symptoms include febrile headache, myalgia (muscle pain), vomiting and jaundice. Examples of some of the other diseases caused by flavivirus species include Japanese encephalitis, Kyasanur Forest disease, Murray Valley encephalitis, Omsk hemorrhagic fever, St. Louis encephalitis and West Nile fever. The mortality rate of these diseases ranges from 0-20%. These diseases share many of the same symptoms, which may include headache, myalgia, fever, hemorrhage, encephalitis, paralysis and rash (“The Springer Index of Viruses,” pgs. 306-319, Tidona and Darai eds., 2001, Springer, N.Y).
D. Bunyaviridae
Bunyaviridae is a family of viruses that includes the genera bunyavirus, phlebovirus, nairovirus, hantavirus and tospovirus. Viruses in three of these genera, hantavirus, phlebovirus and nairovirus, are known to cause VHFs. Members of the Bunyaviridae family are enveloped viruses with a genome that consists of 3 single-stranded RNA segments of negative polarity. The genome segments are designated S (small), M (medium) and L (large). The S segment encodes the nucleocapsid protein (N). The two viral glycoproteins (G1 and G2) are encoded by the M segment and the L segment encodes the viral RNA-dependent RNA polymerase (L). For some Bunyaviridae species, additional viral non-structural proteins are encoded by the S and/or M segment (“The Springer Index of Viruses,” pgs. 141-174, Tidona and Darai eds., 2001, Springer, N.Y.).
Members of the hantavirus genus include, Hantaan virus, Seoul virus, Dobrava-Belgrade virus, Thailand virus, Puumala virus, Prospect Hill virus, Tula virus, Khabarovsk virus, Topografov virus, Isla Vista virus, Sin Nombre virus, New York virus, Black Creek virus, Bayou virus, Caño Delgadito virus, Rio Mamore virus, Laguna Negra virus, Muleshoe virus, El Moro Canyon virus, Rio Segundo virus, Andes virus and Thottapalayam virus. Hantaviruses have a wide geographic distribution and typically cause either hemorrhagic fever with renal syndrome (HFRS) or hantavirus pulmonary syndrome (HPS). Symptoms of HFRS include fever, hemorrhage and renal damage, with a mortality rate up to 15%, depending on the hantavirus species. The first documented case of HFRS occurred in 1934 with a notable epidemic among United Nations soldiers during the Korean War (1951). However, the causative agent of HFRS, Hantaan virus, was not isolated until 1978 (Lee et al. J. Inf. Dis., 1978, 137, 298-308). Symptoms of HPS include fever, pulmonary edema, shock and interstitial pneumonitis (a type of pneumonia involving connective tissue). Sin Nombre virus and Andes virus are two of the hantaviruses that cause a severe form HPS, with an approximately 40% mortality rate. A significant outbreak of pulmonary syndrome occurred in the Southwestern United States in 1993. The etiologic agent of the outbreak was later identified as a hantavirus (Sin Nombre) (Nichol et al. Science, 1993, 262,914-917). The typical route of transmission for hantaviruses is through rodent excreta aerosols, however, Andes virus has been associated with person-to-person transmission (“The Springer Index of Viruses,” pgs. 141-174, Tidona and Darai eds., 2001, Springer, N.Y.; Wells et al. Emerg. Infect. Dis., 1997, 3, 171-174).
Members of the phlebovirus genus include Bujaru virus, Chandiru virus, Chilibre virus, Frijoles virus, Punta Toro virus, Rift Valley Fever virus, Salehebad virus, Sandfly fever Naples virus, Uukuniemi virus, Aguacate virus, Anhanga virus, Arboledas virus, Arumowot virus, Caimito virus, Chagres virus, Corfou virus, Gabek Forest virus, Gordil virus, Itaporanga virus, Odrenisrou virus, Pacui virus, Rio Grande virus, Sandfly fever Sicilian virus, Saint-Floris virus and Urucuri virus. Several phleboviruses (e.g., Sandfly fever Naples virus, Sandfly fever Sicilian virus, Chandiru virus and Chagres virus) cause phlebotomus fever, which is typically found in America and the Mediterranean region. Phlebotomus fever, a non-fatal disease, is transmitted by phlebotomines (sand flies) and induces fever, myalgia (muscle pain) and other flu-like symptoms. Rift Valley fever virus, transmitted by mosquitoes, causes a disease of the same name in Africa. Rift Valley fever is characterized by hemorrhagic fever, hepatitis and encephalitis.
Members of the nairovirus genus include Crimean-Congo hemorrhagic fever virus, Dera Ghazi Khan virus, Dugbe virus, Hughes virus, Nairobi sheep disease virus, Qalyub virus, Sakhalin virus and Thiafora virus. Nairoviruses are primarily found in Africa, Asia, Europe and the Middle East. In humans, nairoviruses can cause hemorrhagic fever (Crimean-Congo hemorrhagic fever), Nairobi sheep disease and Dugbe disease. Nairoviruses are typically transmitted to humans by ticks. The first recognized description of Crimean-Congo hemorrhagic fever dates back to the year 1110. This disease is characterized by sudden onset of fever, nausea, severe headache, myalgia and hemorrhage. The mortality rate is approximately 30%. Nairobi sheep disease symptoms include fever, joint pains and general malaise, while Dugbe disease results in fever and prolonged thrombocytopenia (abnormal reduction in platelets) (“The Springer Index of Viruses,” pgs. 141-174, Tidona and Darai eds., 2001, Springer, N.Y.).
E. Arenaviruses
Arenavirus is the sole genus of the family Arenaviridae. Arenaviruses are enveloped viruses with a genome that consists of 2 single-stranded RNA segments of negative polarity. The negative-sense RNA of the arenavirus genome serves as both a template for transcription of complementary RNA as well as a template for protein synthesis (ambisense RNA). The genome segments are designated S, which encodes the nucleocapsid protein (NP) and the precursor glycoprotein (GPC), and L, which encodes the zinc-binding protein (Z) and the RNA-dependent RNA polymerase (L).
Members of the arenavirus genus include lymphocytic choriomeningitis virus (LCMV), Lassa virus, Ippy virus, Mobala virus, Mopeia virus, Amapari virus, Flexal virus, Guanarito virus, Junin virus, Latino virus, Machupo virus, Parana virus, Pichinde virus, Pirital virus, Oliveros virus, Sabia virus, Tacaribe virus, Tamiami virus, Whitewater Arroyo virus and Pampa virus. A number of arenaviruses are known to cause disease in humans, including LCMV, Lassa virus, Junin virus, Machupo virus, Guanarito virus and Sabia virus. LCMV has a world-wide geographic distribution and infection with LCMV leads to fever, malaise, weakness, myalgia and severe headache. The remaining disease-causing arenaviruses are more limited in their distribution. Lassa fever is found in West Africa and is characterized by fever, headache, dry cough, exudative pharyngitis and hemorrhage. Sabia fever is found is Brazil with symptoms including fever, headache, myalgia (muscle pain), nausea, vomiting and hemorrhage. Junin virus, Machupo virus and Guanarito virus are the causative agents of Argentinean hemorrhagic fever, Bolivian hemorrhagic fever and Venezuelan hemorrhagic fever, respectively, and as their names suggest, are found only in Argentina, Bolivia and Venezuela. Symptoms of these hemorrhagic fevers include malaise, fever, headache, arthralgia (joint pain), nausea, vomiting, hemorrhage and CNS involvement (“The Springer Index of Viruses,” pgs. 36-42, Tidona and Darai eds., 2001, Springer, N.Y).
F. Bioagent Detection
A problem in determining the cause of a natural infectious outbreak or a bioterrorist attack is the sheer variety of organisms that can cause human disease. There are over 1400 organisms infectious to humans; many of these have the potential to emerge suddenly in a natural epidemic or to be used in a malicious attack by bioterrorists (Taylor et al., Philos. Trans. R. Soc. London B. Biol. Sci., 2001, 356, 983-989). This number does not include numerous strain variants, bioengineered versions, or pathogens that infect plants or animals.
Much of the new technology being developed for detection of biological weapons incorporates a polymerase chain reaction (PCR) step based upon the use of highly specific primers and probes designed to selectively detect individual pathogenic organisms. Although this approach is appropriate for the most obvious bioterrorist organisms, like smallpox and anthrax, experience has shown that it is very difficult to predict which of hundreds of possible pathogenic organisms might be employed in a terrorist attack. Likewise, naturally emerging human disease that has caused devastating consequence in public health has come from unexpected families of bacteria, viruses, fungi, or protozoa. Plants and animals also have their natural burden of infectious disease agents and there are equally important biosafety and security concerns for agriculture.
An alternative to single-agent tests is to do broad-range consensus priming of a gene target conserved across groups of bioagents. Broad-range priming has the potential to generate amplification products across entire genera, families, or, as with bacteria, an entire domain of life. This strategy has been successfully employed using consensus 16S ribosomal RNA primers for determining bacterial diversity, both in environmental samples (Schmidt et al., J. Bact., 1991, 173, 4371-4378) and in natural human flora (Kroes et al., Proc Nat Acad Sci (USA), 1999, 96, 14547-14552). The drawback of this approach for unknown bioagent detection and epidemiology is that analysis of the PCR products requires the cloning and sequencing of hundreds to thousands of colonies per sample, which is impractical to perform rapidly or on a large number of samples.
Conservation of sequence is not as universal for viruses, however, large groups of viral species share conserved protein-coding regions, such as regions encoding viral polymerases or helicases. Like bacteria, consensus priming has also been described for detection of several viral families, including coronaviruses (Stephensen et al., Vir. Res., 1999, 60, 181-189), enteroviruses (Oberste et al., J. Virol., 2002, 76, 1244-51); Oberste et al., J. Clin. Virol., 2003, 26, 375-7); Oberste et al., Virus Res., 2003, 91, 241-8), retroid viruses (Mack et al., Proc. Natl. Acad. Sci. U.S.A., 1988, 85, 6977-81); Seifarth et al., AIDS Res. Hum. Retroviruses, 2000, 16, 721-729); Donehower et al., J. Vir. Methods, 1990, 28, 33-46), and adenoviruses (Echavarria et al., J. Clin. Micro., 1998, 36, 3323-3326). However, as with bacteria, there is no adequate analytical method other than sequencing to identify the viral bioagent present.
In contrast to PCR-based methods, mass spectrometry provides detailed information about the molecules being analyzed, including high mass accuracy. It is also a process that can be easily automated. DNA chips with specific probes can only determine the presence or absence of specifically anticipated organisms. Because there are hundreds of thousands of species of benign pathogens, some very similar in sequence to threat organisms, even arrays with 10,000 probes lack the breadth needed to identify a particular organism.
There is a need for a method for identification of bioagents which is both specific and rapid, and in which no culture or nucleic acid sequencing is required. Disclosed in U.S. Patent Application Publication Nos. 2003-0027135, 2003-0082539, 2003-0228571, 2004-0209260, 2004-0219517 and 2004-0180328, and in U.S. application Ser. Nos. 10/660,997, 10/728,486, 10/754,415 and 10/829,826, all of which are commonly owned and incorporated herein by reference in their entirety, are methods for identification of bioagents (any organism, cell, or virus, living or dead, or a nucleic acid derived from such an organism, cell or virus) in an unbiased manner by molecular mass and base composition analysis of “bioagent identifying amplicons” which are obtained by amplification of segments of essential and conserved genes which are involved in, for example, translation, replication, recombination and repair, transcription, nucleotide metabolism, amino acid metabolism, lipid metabolism, energy generation, uptake, secretion and the like. Examples of these proteins include, but are not limited to, ribosomal RNAs, ribosomal proteins, DNA and RNA polymerases, RNA-dependent RNA polymerases, RNA capping and methylation enzymes, elongation factors, tRNA synthetases, protein chain initiation factors, heat shock protein groEL, phosphoglycerate kinase, NADH dehydrogenase, DNA ligases, DNA gyrases and DNA topoisomerases, helicases, metabolic enzymes, and the like.
To obtain bioagent identifying amplicons, primers are selected to hybridize to conserved sequence regions which bracket variable sequence regions to yield a segment of nucleic acid which can be amplified and which is amenable to methods of molecular mass analysis. The variable sequence regions provide the variability of molecular mass which is used for bioagent identification. Upon amplification by PCR or other amplification methods with the specifically chosen primers, an amplification product that represents a bioagent identifying amplicon is obtained. The molecular mass of the amplification product, obtained by mass spectrometry for example, provides the means to uniquely identify the bioagent without a requirement for prior knowledge of the possible identity of the bioagent. The molecular mass of the amplification product or the corresponding base composition (which can be calculated from the molecular mass of the amplification product) is compared with a database of molecular masses or base compositions and a match indicates the identity of the bioagent. Furthermore, the method can be applied to rapid parallel analyses (for example, in a multi-well plate format) the results of which can be employed in a triangulation identification strategy which is amenable to rapid throughput and does not require nucleic acid sequencing of the amplified target sequence for bioagent identification.
The result of determination of a previously unknown base composition of a previously unknown bioagent (for example, a newly evolved and heretofore unobserved virus) has downstream utility by providing new bioagent indexing information with which to populate base composition databases. The process of subsequent bioagent identification analyses is thus greatly improved as more base composition data for bioagent identifying amplicons becomes available.
The present invention provides, inter alia, methods of identifying unknown viruses, including viruses of the Filoviridae, Flaviviridae, Bunyaviridae and Arenaviridae families. Also provided are oligonucleotide primers, compositions and kits containing the oligonucleotide primers, which define viral bioagent identifying amplicons and, upon amplification, produce corresponding amplification products whose molecular masses provide the means to identify viruses of the Filoviridae, Flaviviridae, Bunyaviridae and Arenaviridae families at the sub-species level.